Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar;182(3):1326-1345.
doi: 10.1104/pp.19.01255. Epub 2019 Dec 11.

Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings

Franziska K Kretzschmar  1 , Nathan M Doner  2 , Hannah E Krawczyk  1 , Patricia Scholz  1 , Kerstin Schmitt  3 , Oliver Valerius  3 , Gerhard H Braus  3 , Robert T Mullen  2 , Till Ischebeck  4
Affiliations

Identification of Low-Abundance Lipid Droplet Proteins in Seeds and Seedlings

Franziska K Kretzschmar et al. Plant Physiol. 2020 Mar.

Abstract

The developmental program of seed formation, germination, and early seedling growth requires not only tight regulation of cell division and metabolism, but also concerted control of the structure and function of organelles, which relies on specific changes in their protein composition. Of particular interest is the switch from heterotrophic to photoautotrophic seedling growth, for which cytoplasmic lipid droplets (LDs) play a critical role as depots for energy-rich storage lipids. Here, we present the results of a bottom-up proteomics study analyzing the total protein fractions and LD-enriched fractions in eight different developmental phases during silique (seed) development, seed germination, and seedling establishment in Arabidopsis (Arabidopsis thaliana). The quantitative analysis of the LD proteome using LD-enrichment factors led to the identification of six previously unidentified and comparably low-abundance LD proteins, each of which was confirmed by intracellular localization studies with fluorescent protein fusions. In addition to these advances in LD protein discovery and the potential insights provided to as yet unexplored aspects in plant LD functions, our data set allowed for a comparative analysis of the LD protein composition throughout the various developmental phases examined. Among the most notable of the alterations in the LD proteome were those during seedling establishment, indicating a switch in the physiological function(s) of LDs after greening of the cotyledons. This work highlights LDs as dynamic organelles with functions beyond lipid storage.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Graphical representation of proteomic data derived from Arabidopsis siliques, seeds, and seedlings. A, Tissues were collected from two developmental stages of silique development (phase I, 7–14 DAF; phase II, 14–21 DAF) and six stages of seed germination and seedling establishment (rehydrated seeds, stratified seeds, and seedlings from 24 to 60 h of growth). B, Venn diagram of the distribution of all detected proteins from each of the different developmental stages examined. Proteins identified with at least two peptides using the iBAQ algorithm were grouped into four groups, as depicted. Common proteins were identified via InteractiVenn (Heberle et al., 2015). C and D, PCA was performed to compare the distribution of the five biological replicates at each developmental stage for both the total protein and LD-enriched fractions. Numbers in brackets give the percentage of the total variance represented by Components 1 and 2, respectively.
Figure 2.
Figure 2.
Identification of protein abundance clusters in total protein fractions derived from Arabidopsis siliques, seeds, and seedlings. A, Hierarchical clustering analysis of the normalized protein abundance over each of the developmental stages examined. In total, 40 row clusters were defined. Clusters harboring >20 proteins are labeled C1–C16. B and C, The expression profiles of all proteins in Clusters 1 and 2 (B; pink and brown lines; seed-specific proteins) and Clusters 11 and 12 (C; red and blue lines; early seedling establishment proteins) are shown. As silique samples also contain proteins derived from the silique wall, they should not necessarily be considered seed precursors, as indicated by the interrupted line between the II and RS samples. I, Phase I siliques; II, phase II siliques; RS, rehydrated seeds; StS, stratified seeds; 24 to 60 h, seedlings 24 to 60 h after stratification.
Figure 3.
Figure 3.
Changes in protein intensity of functional groups, based on GO terms, in the total protein fractions derived from Arabidopsis siliques, seeds, and seedlings. Proteins were assigned to GO terms and the relative abundance (rLFQ) of all proteins within a GO term is shown for each stage examined. “n total” corresponds to the total number of genes assigned to one GO term, “n TF” to the number of proteins detected in the total cellular fraction assigned to this GO term. Darker red represents higher total intensities compared to the other growth stages. Shown are selected GO terms.
Figure 4.
Figure 4.
Enrichment analysis of proteins in the LD-enriched fractions derived from Arabidopsis siliques, seeds, and seedlings. A volcano plot was constructed to visualize proteins that are significantly LD enriched (upper right). The developmental stage (siliques, seeds, and seedlings) with the highest abundance (riBAQ) was chosen for each protein and the log2-transformed values and P-values were calculated at this stage. Only proteins detected in four of the five replicates and with an riBAQ >0.1 are included in this figure. As depicted in the legend, known LD and peroxisomal proteins are indicated in blue and orange, respectively, and the proteins chosen for further study that did or did not localize to LDs in pollen tubes and tobacco leaves are shown in blue and green, respectively. Black lines indicate a false discovery rate of 0.001. Abbreviations: ATS3A, EMBRYO-SPECIFIC PROTEIN; BCSAP, BRISC COMPLEX SUBUNIT ABRO1-LIKE PROTEIN; CCLP, CURCULIN-LIKE LECTIN FAMILY PROTEIN; SMT, STEROL METHYLTRANSFERASE; TMPU, TRANSMEMBRANE PROTEIN OF UNKNOWN FUNCTION.
Figure 5.
Figure 5.
Subcellular localization of selected candidate LD proteins in N. tabacum pollen tubes. A to F, Candidate proteins fused to mVenus at their C termini were transiently expressed in N. tabacum pollen tubes (cyan channel). LDs were stained with Nile red (magenta channel). In the merge channel, colocalization appears white. Note, in B, that expression of LDDH1-mVenus led to clustering of LDs. Bars = 10 μm.
Figure 6.
Figure 6.
Subcellular localization of selected LD protein candidates in N. benthamiana leaves. A to F, Candidate proteins fused to mCherry at their N or C termini were transiently expressed in N. benthamiana leaves (cyan channel). LDs were stained with BODIPY 493/503 (magenta channel). In the corresponding merge channel, note that a torus fluorescence pattern attributable to the expressed fusion protein encircles the BODIPY-stained LDs. Boxes denote portions of the cells shown at higher magnification in the insets. Bars = 10 μm (1 μm in insets).
Figure 7.
Figure 7.
Subcellular localization of SEC61γ in tobacco pollen tubes and N. benthamiana leaves. SEC61γ fused at its C terminus to mVenus or mCherry was transiently expressed in N. tabacum pollen tubes (A and B) or in N. benthamiana leaves (C). LDs were stained with Nile red (A and B) or BODIPY 493/503 (C and D). SEC61γ was cotransformed with the ER marker ERD2-CFP (A and B) and partially colocalizes with the ER. SEC61γ also accumulates at potential ER-LD contact sites (B–D). The box in C indicates the area of the cells shown at higher magnification in D. Bars = 10 μm (A and C) or 2 μm (B and D).
Figure 8.
Figure 8.
Dynamic composition of the LD proteome derived from Arabidopsis siliques, seeds, and seedlings. The riBAQ intensities of LD-associated proteins in the LD-enriched fraction were calculated as a percentage of the riBAQ of all known LD-associated proteins. This way, the contribution of each protein to the complete LD proteome and the dynamic changes in the abundance of the LD proteins can be observed. Protein isoform numbers separated by a slash indicate that these proteins could not be distinguished based on the proteomic data. Two highly similar genes of the steroleosin family are both annotated as HSD1. I, Phase I siliques; II, phase II siliques; RS, rehydrated seeds; StS, stratified seeds; 24 to 60 h, seedlings 24 to 60 h after stratification. As silique samples also contain proteins derived from silique wall, they should not necessarily be considered to be seed precursors, as indicated by the interrupted line between the II and RS samples. n = 5 per stage. Error bars represent the sd.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arsova B, Watt M, Usadel B (2018) Monitoring of plant protein post-translational modifications using targeted proteomics. Front Plant Sci 9: 1168. - PMC - PubMed
    1. Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein SD, Daum G (1999) Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J Bacteriol 181: 6441–6448 - PMC - PubMed
    1. Baud S, Boutin JP, Miquel M, Lepiniec L, Rochat C (2002) An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40: 151–160
    1. Baud S, Dichow NR, Kelemen Z, d’Andréa S, To A, Berger N, Canonge M, Kronenberger J, Viterbo D, Dubreucq B, et al. (2009) Regulation of HSD1 in seeds of Arabidopsis thaliana. Plant Cell Physiol 50: 1463–1478 - PubMed
    1. Beaudoin F, Wilkinson BM, Stirling CJ, Napier JA (2000) In vivo targeting of a sunflower oil body protein in yeast secretory (sec) mutants. Plant J 23: 159–170 - PubMed

Publication types

MeSH terms